Mechanisms of the anti-angiogenic effects of microtubule binding anti-cancer drugs

Paclitaxel is a microtubule stabiliser widely used in cancer treatment. In addition to its cytotoxic effect, paclitaxel inhibits angiogenesis at subtoxic doses. This anti-angiogenic effect is thought to be mediated through cytostasis of endothelial cells or altered microtubule dynamics. There is also evidence that more complex processes may be involved. Understanding the underlying molecular events is crucial for the use of paclitaxel in anti-angiogenic therapy. Since the small GTPase RhoA is essential for angiogenesis and microtubule dynamics, this study proposed RhoA as the key determinant of paclitaxel-induced anti-angiogenesis.A clear separation of the cytotoxic and anti-angiogenic effects of paclitaxel was facilitated by the 24 h differential cytotoxicity assay against 1A9 ovarian carcinoma and human umbilical vein endothelial cells (HUVEC), as well as HUVEC tube formation assay. Paclitaxel inhibited tube formation at subtoxic doses (≤ 10 nM). G-LISA detection of active RhoA in HUVEC showed that paclitaxel inhibited RhoA activation and this was strongly correlated to its anti-angiogenicity. Transient transfection of HUVEC with active RhoA mutant elicited attenuation of the anti-angiogenic effect of paclitaxel. Similarly, the anti-angiogenicity of paclitaxel was attenuated by lysophosphatidic acid, a RhoA activator. RhoA inactivation is required for inhibition of angiogenesis by paclitaxel.The mechanisms behind these observations might involve interaction between p27Kip1 (cyclin-dependent kinase inhibitor) and stathmin (microtubule depolimerisation protein). Western blot analysis indicated augmented ratios of inactive, phosphorylated stathmin(serines 16 and 63) to total stathmin at 10 nM paclitaxel. Co-immunoprecipitation of stathmin and p27Kip1 showed an increase in stathmin-p27Kip1 binding. The RhoA-dependent anti-angiogenic effect of paclitaxel is mediated through stathmin phosphorylation and increased stathmin-p27Kip1 interaction.Non-microtubule targeting agents were also included in this study. AG1478 and cisplatin yielded anti-angiogenic effects which did not require RhoA inactivation. Gemcitabine, which was not anti-angiogenic, inhibited tube formation under conditions in which RhoA was activated. The RhoA-dependent inhibition of angiogenesis could be microtubule-dependent.The combination use of paclitaxel with therapeutic agents that target RhoA, stathmin and p27Kip1 may enhance its application in anti-angiogenic therapy